Methods for detecting microorganisms in a clinical sample generally comprise the following steps:
1) Optionally treating the sample to promote bacterial growth, the length of this step varying in relation to the type of sample (blood, urine, stools, cerebrospinal fluid, other puncture fluids) and in relation to the bacterial load;
2) Seeding one or more agar growth media which may or may not be selective;
3) Incubating for 18 to 48 h at 37° C.;
4) Observing the different types of colonies obtained (size, appearance, colour . . . );
5) Collecting one or more isolates of each type of colony;
6) Actual identification, if necessary using an automated system or an identification gallery;
7) Obtaining an antibiogram in parallel which requires a minimum time of 16 h, or obtaining an antibiogram consecutively which requires an additional time of 18 h to determine the profile of the microorganism with respect to antibiotics and/or antifungals.
The diagnosis of an infection by microorganisms is therefore generally long, the time notably depending on the type of sample to be analysed. This waiting time is particularly problematic if the sample to be analysed is a blood sample. If it is sought to identify the presence of microorganisms in blood, the first treatment step of the sample by blood culture which allows the detection threshold of the microorganisms to be reached, may take up to 7 days. The duration of this first treatment step cannot be reduced since it is dependent on automated systems. On the slightest sign of culture positivity, Gram staining is performed on a sample taken by aseptic puncture of the film using a sterile syringe. This direct examination will allow recognition of the morphology of the bacterial agent present in the bottle which may guide towards identification of the germ. The information is then immediately transmitted to the clinician since it may allow the start of an antibiotic treatment that has not yet been initiated or the correction of probabilistic antibiotherapy. Nevertheless, no precise characterization of the microorganism under consideration is possible at this level, and even less so a profile of particular resistance. It is therefore particularly useful to be subsequently able to identify rapidly the microorganisms present in the biological sample together with their possible resistance phenotypes so as to initiate or rectify antibiotic treatment.
Different types of microorganisms can be present in positive blood cultures. It is a fact that staphylococci (not only Staphylococcus aureus but also negative coagulase staphylococci) are regularly found. Gram-negative bacilli especially Escherichia coli (and more generally enterobacteriaceae), anaerobic bacteria of the colon, members of the groups Klebsiella/Enterobacter, Pseudomonas aeruginosa, Proteus spp and Providencia spp, have been detected for example after traumatic injury or after surgery on an already contaminated body region. The detection of salmonella in the blood of individuals suffering from systemic salmonellosis is not unusual. Numerous other microbial genii have been found in cultures of blood samples such as streptococci, enterococci, Brucella, Pasteurella, pneumococci, Neisseria, Listeria, Clostridium, corynebacteria, Bacteroids, bacteria in the hacek group but also yeasts and parasites.
Different techniques are used for precise identification of these microorganisms and their possible resistance phenotypes, but the results are generally only obtained after a relatively long period.
Samples are coated on a medium containing fresh blood and chocolate blood agar to which other media may be added depending on the results of microscopy examinations.
For each positive bottle, the following are carried out:
determining the morphology of the colonies,
oxidase and catalase testing,
biochemical and antigenic identification to identify the microorganism,
susceptibility tests, evaluation of Minimum Inhibitory Concentrations (MIC).
It has been shown that it is possible to obtain an antibiogram directly from a positive blood culture using an inoculum equivalent to 0.5 McF from the blood culture broth. A Mueller-Hinton agar is inoculated using the <<flooding>> technique. The correlation with the standard antibiogram is apparently found in 95% of cases. The result appears no earlier than 16 h after evidencing of the positive blood culture (Antibiotics In Laboratory Medicine, Victor Lorian, M.D. Editor, 5th Edition; Doern et al., 1981, Antimicrob Agents Chemother. 20(5): 696-698. Antimicrobial Agents).
There is therefore a major need for new, more rapid methods for detecting microorganisms, and/or their possible associated resistances, in a biological sample, these methods having to maintain satisfactory susceptibility and specificity.
β-lactams represent the largest family of antibiotics on account of the large number of molecules belonging thereto, and on account of their associated pharmacological properties and spectra which allow the combating of most bacterial species. β-lactams are rightly the antibiotics that are the most prescribed in general medicine. Their spectrum of activity varies as a function of their class (penicillins or cephalosporins) and at times as a function of the molecules in each class. For example, the G and V penicillins are rather more active on Gram-positive cocci and anaerobic bacteria, whilst the spectrum of aminopenicillins extends to a few Gram-negative bacilli. The addition of a β-lactamase inhibitor also leads to observing activity on some bacteria producing these β-lactamases. Cephalosporins further extend the spectrum of activity to Gram-negative bacilli but are a little less active on Gram-positive cocci.
Among the β-lactams, cephalosporins account for the category of antibiotics that is most often prescribed. The frequent use of cephalosporins has led to the spreading of strains resistant to these molecules. These strains in particular manage to survive the pressure of cephalosporin selection through β-lactamase activity. The detection of combined resistances against 1st, 2nd and 3rd generation (C3G) cephalosporins in Enterobacteriaceae is becoming of major therapeutic importance. The increasing resistance of Enterobacteriaceae to antibiotics is of world concern with an increasing impact of extended spectrum β-lactamases (ESBLs), spreading in the community in particular. In 2002, less than 1% of Enterobacteriaceae strains produced an ESBL. In 2006, they represented 1 to 5% of strains.
The resistance to C3Gs whether due to ESBLs or to other β-lactamases (cephalosporinase or carbapenemase) is therefore on the increase and has become significant in particular for Escherichia coli and Klebsiella pneumoniae. The emergence of these enzymes has recently been reported in non-fermenting Gram-negative bacilli such as Acinetobacter baumannii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. It is therefore of increasingly greater importance to be able to detect these ESBLs.
The detection of ESBLs in Enterobacteriaceae is simple to perform in most cases; it is implemented by synergy between a mixture of [amoxicillin+clavulanic acid (AMC)]and a C3G (ceftazidime is the most susceptible) or aztreonam. Nonetheless, obtaining a result requires much time. The synergy test is performed by arranging discs of AMC and the chosen C3G (or aztreonam) at a distance of 30 mm away from each other, centre to centre. The detection of ESBLs on the other hand is more difficult for strains which are also hyper-productive of cephalosporinase (AmpC) such as Enterobacter. In this latter case, it is easier to visualize the synergy between AMC and cefepime or cefpirome. Finally, in some species such as Proteus mirabilis, Proteus vulgaris, Proteus penneri, Morganella morganii, Providencia stuartii and Providencia rettgeri, ESBLs are weakly expressed and are therefore even more difficult to detect. In these cases, the synergy test is optimized by placing the discs at a distance of 40-45 mm, instead of 30 mm, following CA-SFM recommendations (Comité de l'antibiogramme de la Société française de microbiologie) in 2007. Automated bacteriological systems for identification and antibiograms are increasingly more used (Mini-Api® marketed by bioMérieux Clinical Diagnostics, Phoenix® marketed by BD Diagnostics, MicroScan® marketed by Siemens, Vitek®, Vitek-2® and Vitek Compact® marketed by bioMérieux). In general, these automated systems detect ESBLs relatively well in strains which do not usually hyper-produce cephalosporinases (E. coli, K. pneumoniae, K. oxytoca). For the other species (Enterobacter, Citrobacter, Serratia, etc.), their susceptibility and especially their specificity are not quite as good as those of manual methods. Additional tests prove to be necessary.
The detection of strains resistant to C3Gs may therefore be easy for some resistances related to some species, but in general no procedure has 100% sensitivity or allows a rapid result to be obtained.
Numerous improvements have been proposed to accelerate the process of detecting and identifying microorganisms present in blood cultures in particular. First, the improvements in culture media and growth techniques have reduced culture times. The latest generation of automated machines even allows the detection of small bacterial growth. Independently of conventional techniques, when growth is detected by an automated system, it is also possible to perform direct identification of bacteria by molecular biology (amplification and sequencing, FISH, DNA chips or specific probes . . . ). Nonetheless, most of these systems are usually not open systems and only allow the detection of one or of a small number of specific microorganisms. In addition, they possibly may not provide information on susceptibility or presumed resistance against an antibiotic. Also, these methods are efficient but expensive and/or require highly qualified laboratory technicians.
Attempts to accelerate more specific detection of microorganisms resistant to a family of antibiotics have been described. For example, Weinbren and Borthwick (Weinbren and Borthwick, 2005, J. Antimicrob. Chemother. 55:131-132) have described a method for the non-chromogenic detection of ESBL-producing microorganisms, in a blood culture whereby a sample of blood culture is taken and transferred onto an agar medium, discs of cefpodoxime and cefpodoxime-clavulanate then being applied to the medium. Nevertheless, this culture step on an agar medium requires an additional minimum time wait of 3.5 h to 6 h before it is possible to obtain initial results after the completion of blood culture treatment.
Navon-Venezia et al. (Navon-Venezia et al., 2005, J. Clin. Microb. 43:439-441) have described a method for detecting ESBL-producing bacteria from positive blood cultures consisting of taking a sample of blood culture broth and seeding it directly onto a Mueller-Hinton medium containing discs of cefpodoxime and cefpodoxime+clavulanate, without including an isolation step on agar medium. No chromogenic agent is used in this method which necessitates a culture step of 16 h to 18 h following the recommendations of the American reference work CLSI/NCCLS (Clinical and Laboratory Standards Institute/National Committee on Clinical Laboratory Standards).
Chapin and Musgnug (2003) (Chapin and Musgnug, 2003, J. Clin Microb. 41:4751-4754) have described a direct method for testing susceptibility to antimicrobials on positive blood cultures consisting of centrifuging 10 ml of inoculated blood culture in a serum separating tube containing a gel. The microorganisms remaining on the surface of the gel are then collected and replaced in suspension to inoculate microplates containing different dilutions of specific antibiotics. The result is only obtained however 18 to 24 h after inoculation.
Chen et al. (2008) (Chen et al., 2008, J. Microbiol. Immunol. Infect. 41:259-264) have described a method for identifying the susceptibility of microorganisms to a group of antibiotics using positive blood cultures which entails taking a sample of positive blood culture, removing the red blood cells by lysis and centrifugation, placing in suspension the bacterial residue in a saline solution and testing using a Vitek-2® system or AST (antimicrobial susceptibility testing). It is nevertheless necessary to wait between 2 h30 and 16 h15 after loading the Vitek-2® or AST system before it is possible to observe initial results.
The team Jain et al. (Jain et al., 2007, J. Antimicrob. Chemother. 60:652-654) has also described a relatively rapid method for detecting extended spectrum β-lactamases (ESBLs) by colour change of a supernatant of blood culture broth after centrifugation. On account of haemolysis problems interfering with the change in colour of the chromogenic agent, the authors proposed the prior conducting of a sub-culture in a fresh medium, thereby lengthening the time of the test method.
These techniques whereby resistant microorganisms are detected after a sub-culture step therefore remain relatively lengthy techniques.